The present invention relates to microarray spotting instruments. More particularly, the present invention relates to improved spotting instruments that incorporate sensors and methods of using those sensors for improving performance of the spotting instruments.
As is well known (and described for example in U.S. Pat. No. 5,807,522 to Brown et al. and in xe2x80x9cDNA Microarrays: A Practical Approachxe2x80x9d, Schena, Mark, New York, Oxford University Press, 1999, ISBN 0-19-963776-8), microarrays are arrays of very small samples of purified DNA or protein target material arranged as a grid of hundreds or thousands of small spots on a solid substrate. When the microarray is exposed to selected probe material, the probe material selectively binds to the target spots only where complementary bonding sites occur, through a process called hybridization. Subsequent quantitative scanning by a fluorescent microarray scanner (i.e., a scanning instrument) may be used to produce a pixel map of fluorescent intensities (See, e.g., U.S. Pat. No. 5,895,915, to DeWeerd et al.). This fluorescent intensity map can then be analyzed by special purpose quantitation algorithms which reveal the relative concentrations of the fluorescent probes and hence the level of gene expression, protein concentration, etc., present in the cells from which the probe samples were extracted.
The microarray substrate is generally made of glass which has been treated chemically to provide for molecular attachment of the spot samples of microarray target material. The microarray substrate is also generally of the same size and shape as a standard microscope slide, about 25 mmxc3x9775 mmxc3x971 mm thick. The array area can extend to within about 1.5 mm of the edges of the substrate, or can be smaller. The spots of target material (typically DNA) are approximately round. The spot diameter is generally determined by the dispensing or spotting technique used and typically varies from about 75 microns to about 500 microns, and may be as small as about 20 microns. The general trend is toward smaller spots, which produce more compact arrays. The center-to-center spacing between the spots usually falls into the range of 1.5 to 2.5 spot diameters.
FIG. 1A, which is not drawn to scale, shows a top view of a prior art microarray 100. In FIG. 1A, each of the circles represents a tiny spot of target material that has been deposited onto a rectangular glass substrate 101, and the spots are shown in a magnified view as compared to the substrate 101. Assuming typical dimensions of 100 xcexcm spot diameter and 200 xcexcm center-to-center spacing between the spots, the illustrated six by six array of spots covers only a 1100 xcexcm by 1100 xcexcm square area of the 25 mm by 75 mm area defined by the substrate 101. Thousands of spots are usually deposited in a typical microarray and the spots may cover nearly the entire substrate. The portion of the microarray that is covered with spots of target material may be referred to as the xe2x80x9cactive areaxe2x80x9d of the microarray.
There are several well known methods of depositing the spots onto the substrate of a microarray, and instruments that deposit the spots are typically referred to as xe2x80x9cspotting instrumentsxe2x80x9d. One popular method is to use one or more xe2x80x9cpinsxe2x80x9d to transfer the target material from a reservoir onto the microarray substrate. FIG. 1B shows an example of such a prior art pin 102, which includes a pin head 104 and a shaft 106. Both the pin head 104 and the shaft 106 are generally cylindrical, and the pin head 104 and shaft 106 are generally disposed so that they are coaxial. The diameter of the pin head 104 is greater than the diameter of the shaft 106, and the shaft is substantially longer than the pin. One end 107 of the shaft 106 is tapered or sharpened, and the other end of the shaft is attached or bonded to the pin head 104. Examples of such pins are described in, for example, U.S. Pat. No. 5,770,151 (Roach et al.) and U.S. Pat. No. 5,807,522 (Brown et al.).
In operation, the sharp ends 107 of the pins are dipped into a reservoir of the liquid target material so that some of the material is xe2x80x9ccollected byxe2x80x9d or becomes attached to the pins. The sharp ends of the pins are then placed in contact with the substrate to deposit tiny amounts of the material onto selected locations of the substrate. The pins are normally moved by a mechanical or robotic apparatus so the spots may be accurately placed at desired locations on the substrate.
Some types of pins are capable of collecting only enough target material to form a single spot on the microarray before they need to be re-dipped in the reservoir, whereas others can collect enough target material from the reservoir to form several or even hundreds of spots before they need to be re-dipped in the reservoir. In either case, the pins must be manufactured to very precise tolerances to insure that each spot formed by the pin will be of controlled size. As a result of these demanding specifications, the pins are rather expensive (e.g., a single pin typically costs several hundred dollars). Also, the sharp ends of the pins are so small and precisely shaped (e.g., a square tip measuring 50 microns on a side) that the pins are fragile. Accordingly, to prevent damage, the sharp ends of the pins should only be subjected to a tiny force when the sharp ends are placed in contact with the substrate or any other solid object.
Spotting instruments typically form microarrays in batches. For example, in a single xe2x80x9crunxe2x80x9d, a spotting instrument may form up to one hundred identical microarrays. After forming enough spots of a particular target material to complete the batch of microarrays being spotted, the pins generally need to be washed (to remove any excess liquid target material), and then dried before they can be dipped into another reservoir of target material. So the process of forming microarrays with a xe2x80x9cpin-typexe2x80x9d spotting instrument includes steps of (1) positioning a pin over a reservoir of target material; (2) dipping the sharp end of the pin into the reservoir; (3) withdrawing the sharp end of the pin from the reservoir; (4) moving the pin over a selected location within the active area of a microarray; (5) lowering the pin to bring the sharp end of the pin into contact with the microarray substrate to form a single spot of controlled size at the selected location; (6) raising the pin to separate the sharp end of the pin from the substrate; (7) repeating steps (4), (5), and (6) until the pints supply of target material is exhausted or until the desired number of spots have been placed on the bach of microarrays being produced; (8) washing the pin by either placing the pin in a stream of cleaning solution or by dipping the pin into a reservoir of cleaning solution; and (9) drying the pin. The spotting instrument repeats all of these steps numerous times to form a single microarray.
Since microarrays typically include thousands of spots, using only a single pin to form the microarray would be extremely time consuming. Accordingly, spotting instruments are often capable of simultaneously manipulating several pins. FIGS. 1C, 1D, and 1E show side, top, and perspective, views respectively of a printhead 110 that can simultaneously hold sixteen pins 102. Printhead 110 is a solid block of material, typically metal, that defines an array of sixteen apertures 112. The apertures 112 are slightly larger than the outer diameter of the shafts 106 so the shafts can extend through the apertures 112. The apertures 112 are also smaller than the outer diameter of the pin heads 104 so that when the shaft of a pin is dropped into one of the apertures 112, the pin head 104 will be supported by the upper surface of the printhead 110. The pins are thereby xe2x80x9cslip-fitxe2x80x9d into the apertures of the printhead. FIGS. 1F and 1G show side and top views, respectively, of sixteen pins mounted into printhead 110.
FIG. 1H illustrates printhead 110 being lowered to place the sharp ends of the pins 102 into contact with substrate 101 and thereby simultaneously forming sixteen spots of target material on the substrate. As shown, the printhead is generally lowered about 1 mm further than required to place the sharp ends of the pins in contact with the substrate. The slip-fit allows the upper surface of the printhead to be lowered beneath the bottom of the pin heads without imparting significant force to the sharp ends of the pins. The printhead is preferably lowered sufficiently slowly so that the force applied to the sharp ends of the pins (1) is principally determined by the weight of the pin plus a minor additional force due to the friction of the slip-fit and (2) is not significantly affected by inertial forces.
Commercially available printheads provide between 4 and 72 apertures, thereby accommodating between 4 and 72 pins. Commercially available reservoirs provide a plurality of wells, or individual reservoirs, and permit each pin mounted in a printhead to be dipped into a separate well. Two popular reservoirs useful for producing microarrays are the xe2x80x9c96-well platexe2x80x9d and the xe2x80x9c384-well platexe2x80x9d. Each of these plates provides a rectangular array of wells, each well being capable of holding a unique sample of liquid target material. FIG. 1I shows a top view of a 96-well plate. In 96-well plates, the centers of the individual reservoirs are separated by 9.0 mm, and in 384-well plates, the centers of the individual reservoirs are separated by 4.5 mm. The centers of adjacent apertures in commercially available printheads are correspondingly separated by either 9.0 or 4.5 mm.
Jets or nozzles, similar to those used for placing ink onto paper in ink-jet type printers, are another popular device used for forming spots on microarrays. Instead of using pins, jet type spotting instruments use one or more jets to form the spots on the microarray substrate. Each jet generally includes a hollow tube or needle and one end of the tube is configured as a nozzle. Initially, the nozzles are positioned over a reservoir and a vacuum is used to aspirate or collect a few microliters of target material into each of the hollow tubes. The nozzles are then positioned over a microarray substrate and a pulse of pressure applied to the tubes causes each tube to dispense a small amount of target material onto the substrate thereby forming a group of spots. Jet type spotting instruments are similar to pin type instruments. The principal difference is that in jet type instruments, the printhead carries an array of jets instead of an array of pins. In most respects, operation of the two types of instruments is similar.
In general, spotting instruments include mechanisms for holding or manipulating one or more plates (e.g., either 96-well or 384-well), a printhead (e.g., of either the pin-type or the jet-type), a robotic manipulator for controlling the movement of the printhead, mechanisms for holding a plurality of substrates, a pin or jet washer, and a dryer. The act of using a spotting instrument to form spots on a microarray substrate may be referred to as xe2x80x9cprintingxe2x80x9d.
FIG. 2 shows a block diagram illustrating a prior art spotting instrument 200. Instrument 200 includes a processor 210, a position controller 212, a printhead 214, a substrate station 216, and a well station 218. Although not illustrated, it will be appreciated that spotting instrument 200 may additionally include items such as a pin washer and a dryer (or a jet washer and dryer). Printhead 214 may be of the pin-type (i.e., one that holds one or more pins) or of the jet-type (i.e., one that holds one or more jets). Substrate station 216 generally includes platforms or holders for holding several (e.g., one hundred) microarray substrates and may further including a substrate handling system for automatically loading and unloading the substrates. Similarly, well station 218 generally includes platforms or holders for holding several reservoirs of target material (e.g., 96-well or 384-well plates) and may further include a handling system for automatically loading and unloading the reservoirs.
In operation, the processor 210 directs the position controller 212 (which is typically implemented as a robotic manipulator) to place the printhead 214 over one of the reservoirs stored in the well station 218 so that the printhead 214 may collect samples of selected target material. The processor then directs the position controller 212 and printhead 214 so as to print spots of the target material on one or more of the substrates being held in the substrate station 216. After all desired printing of that target material, the printhead may be cleaned and then placed over a different reservoir in the well station 218 to collect samples of some other target material. During printing of a batch of microarrays, it may be necessary to replace some of the reservoirs in the well station 218 with reservoirs containing different samples of target material.
For an experiment with a microarray to be useful, it is important to know the type of target material that was used to print every spot on the microarray. This can be extremely difficult for several reasons. For example, there are typically thousands of spots on a single microarray, and all spots tend to look alike. This makes it difficult to distinguish one spot from another on a single microarray and also makes it difficult to distinguish one microarray from another. Also, there is generally no easily discernable relationship between the location of a particular target material on a microarray (i.e., the location of spots printed using a particular target material) and the location of that target material in one or more of the reservoirs. This is because, for example, spots of different target material are typically printed next to one another and because multiple plates are typically used to form a single microarray.
Several attempts have been made in the prior art to facilitate use of microarrays and to make it easier to discern the type of target material used to form any given spot. For example, bar code labels have been included on the top of microarray substrates. Since the active area of a microarray often covers nearly the entire substrate, such labels are generally extremely small (e.g., 0.9 inches by 0.55 inches), and high-resolution bar code readers are required to read the labels. However, some microarray scanning instruments incorporate such bar code readers and this makes it easier to distinguish one microarray from another.
Another attempt to facilitate use of microarrays has been the inclusion of bar code labels on reservoirs of target material. Some prior art spotting instruments include a fixed bar code reader for reading the labels on the reservoirs. For example, in instrument 200 the well station 218 may include a fixed bar code reader for reading labels on reservoirs as they are moved by the well station""s handling system. Since there is only limited space available on the top of a typical reservoir or plate for placing a bar code label, a convention or standard has developed of placing bar code labels on the sides of plates. In accordance with this convention, spotting instruments that include a bar code reader orient the reader so that it is xe2x80x9csideways lookingxe2x80x9d, or so that light emitted from the reader travels in a direction that is parallel to the ground and perpendicular to the vertical sides of plates. Such fixed, sideways looking, bar code readers are capable of reading bar code labels that are affixed to the sides of the plates in accordance with the prior art convention.
Yet another attempt to facilitate use of microarrays has been the creation of software that allows an operator to specify general characteristics of a desired microarray and that uses those general specifications to generate control signals for controlling a spotting instrument so as to create the desired microarray. For example, such software allows the operator to specify (1) the desired configuration of the spots of a microarray (e.g., the number spots for the microarray, the location of each spot, and the type of target material that should be used for each spot); (2) the number of pins or jets to be used in the printhead; (3) the number and location of plates that will hold all samples of the target material; and (4) the identity of the target material located in each of the wells of each of the plates. The software then uses these general specifications to direct the spotting instrument to form a microarray, or a batch of microarrays, having the desired configuration of spots.
Despite these attempts, there remain significant problems with making and using microarrays. For example, prior art spotting instruments generally rely upon a human operator to verify that the necessary reservoirs or plates are accurately positioned within the instrument""s well station and that substrates are properly mounted in all the holders of the substrate station. If the human operator makes any errors in this regard, the spotting instrument may become damaged or the microarrays may be incorrectly fabricated (e.g., incorrect target material may be used to form some of the spots on the microarrays). The job of the human operator is further complicated because a single xe2x80x9crunxe2x80x9d of a spotting instrument for producing a batch of microarrays generally takes several hours (e.g., twenty hours is a typical figure). Also, during a run, some of the plates must typically be removed from the instrument""s well station and other plates must be added to the well station. If the human operator fails to make these substitutions at the correct times, or fails to notice changes in conditions of the instrument (e.g., a substrate that may have inadvertently become dislodged from one of the holders in the substrate station), the run may be delayed, the spotting instrument may suffer damage, or the run may be wasted since the microarrays may be fabricated incorrectly. The sideways looking bar code readers that have been incorporated into some prior art spotting instruments make the human operator""s job a little easier. However, these bar code readers only inspect the plates while they are in the instrument""s handling system and are not capable of determining whether the plates have been correctly loaded into the holders of the well station, nor do they identify the individual microarray substrates as having been spotted with a particular spotting protocol. Such information would be of value for data tracking and automated operation of downstream microarray processes such as application of probe, hybridization, scanning, quantification, etc.
It would therefore be advantageous to provide methods and apparatus for reducing the need for human supervision of spotting instruments.
These and other objects are provided by incorporating one or more sensors into a spotting instrument. The sensors are preferably mechanically fixed to the printhead and enable the spotting instrument to detect the presence or absence of substrates and/or wells in the spotting instrument.
Still other objects and advantages of the present invention will become readily apparent to those skilled in the art from the following detailed description wherein several embodiments are shown and described, simply by way of illustration of the best mode of the invention. As will be realized, the invention is capable of other and different embodiments, and its several details are capable of modifications in various respects, all without departing from the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not in a restrictive or limiting sense, with the scope of the application being indicated in the claims.